PROJECT SUMMARY MHC class I (MHC-I) -restricted peptide cancer vaccines hold the minimal amount of biochemical information required for generating antigen-specific T cells to elicit anti-tumor responses. On their own, immunization with minimal peptide epitopes does not provide a satisfactory response, so typically long peptides or conjugated delivery systems are necessitated. However, such approaches defeat the directness of short synthetic peptide vaccines. A new peptide vaccine immunization paradigm will be introduced, based on combining (via simple mixing) MHC-I peptide epitopes with a vaccine adjuvant that induces spontaneous nanoliposome-antigen particleization (SNAP). Liposomes that contain small amounts of cobalt porphyrin-phospholipid (CoPoP) rapidly bind short his-tagged peptides (8-9mers) via spontaneous insertion of the his-tag into the bilayer. This gives rise to particleization that is stable in biological media. His-tagged peptides are simply mixed with CoPoP liposomes at the time of vaccination (without further purification) to convert these well-characterized peptides into a 100 nm particle. This approach is potently effective in generating antigen-specific CD8+ T cells. AH1 is a MHC-I H-2Ld model CD8+ epitope derived from the gp70 murine leukemia virus antigen. An AH1-derived peptide can be used with SNAP immunization to generate high numbers of antigen specific CD8+ T cells. SNAP immunization with low nanogram doses peptides completely protects mice from subsequent tumor challenge, and eradicates 100% of lung metastases in a therapeutic vaccine model. Varying components of SNAP immunization will be assessed, including the his-tag length, the density and dose of co-incorporated (MPLA and QS-21; both part of GSK?s vaccine adjuvant AS01) to determine their impact the Ag-specific CD8+ response. Generation of Ag-specific CD44+ CD62L+ cells will be assessed to determine whether such central memory cells are more effective in eradicating tumors. Vaccine efficacy will be tested in multiple prophylactic and therapeutic local and metastasis tumor models. Therapeutic treatment of large tumors will be assessed with the impact of cyclophosphamide and checkpoint blockade. Mechanistic insights will be probed by assessing how the delivered peptide reaches MHC- I. It is hypothesized that his-tag peptides bind to CoPoP liposomes, and undergo serum-stable transit to draining lymph nodes. There, they are phagocytosed by dendritic cells where the reductive environment of phagosomes is also suspected to induce the release of the peptide from the CoPoP liposomes. Toll-like receptor in the bilayer are hypothesized to upregulate the expression of MHC-I within the phagosome. Further studies will assess the viability of SNAP immunization and antigen multiplexing as an in vivo screening tool for peptide microlibraries using established tumor-associated antigens and neoantigens that will be identified with next-generation sequencing. Finally, the safety and cobalt response of the system will be assessed. These studies will provide substantial advancement for short, MHC-I-restricted peptide-based cancer vaccines for cancer therapy and immunogenic epitope screening.